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A kind of cultivating method for improving porcine epidemic diarrhea virus virulence

A technology for porcine epidemic diarrhea and culture method, which is applied in the directions of microorganism-based methods, viruses, culture processes, etc., can solve the problem of low virus virulence, and achieves solving the problem of low virus virulence, increasing effective virus content, and protecting invariance. inactive effect

Active Publication Date: 2021-08-17
SICHUAN HUASHEN ANIMAL BIOLOGICAL PRODS
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Aiming at the above-mentioned deficiencies in the prior art, the present invention provides a cultivation method for increasing the virulence of porcine epidemic diarrhea virus, which can significantly increase the virus content, avoid virus inactivation, and effectively solve the problem of low virulence of the virus

Method used

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  • A kind of cultivating method for improving porcine epidemic diarrhea virus virulence

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Embodiment 1

[0019] A culture method for improving porcine epidemic diarrhea virus virulence, comprising the following steps:

[0020] (1) Vero cells were subcultured in a bottle at a volume ratio of 1:3, and then inserted into 1300 mL of DMEM medium containing 8vt% newborn bovine serum and a pH value of 7.2, and cultured at 36.5°C and 9r / h for 45h. A dense monolayer was obtained;

[0021] (2) pour out the used DMEM medium, then inoculate the porcine epidemic diarrhea virus of 1vt% in the dense monolayer of step (1) gained, cultivate 50min under 36.5 ℃ of temperature, 9r / h rotating speed condition, obtain mixture ;

[0022] (3) Add 1400mL of DMEM medium containing 1vt% newborn calf serum, 8 μg / mL insulin and 7g / L trehalose, and a pH value of 7.2 to the mixture obtained in step (2). Under cultivation, when the lesion rate is greater than 80%, the virus is harvested to obtain porcine epidemic diarrhea virus.

Embodiment 2

[0024] A culture method for improving porcine epidemic diarrhea virus virulence, comprising the following steps:

[0025] (1) The Vero cells were subcultured in a bottle at a volume ratio of 1:3, and then inserted into 1500 mL of DMEM medium containing 10vt% newborn bovine serum and a pH value of 7.3, and cultured for 48 hours at a temperature of 36.5°C and a rotational speed of 10r / h. A dense monolayer was obtained;

[0026] (2) pour out the used DMEM medium, then inoculate the porcine epidemic diarrhea virus of 2vt% in the dense monolayer of step (1) gained, cultivate 60min under 36.5 ℃ of temperature, 10r / h rotating speed condition, obtain mixture ;

[0027] (3) Add 1500mL of DMEM medium containing 2vt% newborn calf serum, 10μg / mL insulin and 8g / L trehalose, and a pH value of 7.3 to the mixture obtained in step (2), at a temperature of 36.5°C and a rotation speed of 10r / h Under cultivation, when the lesion rate is greater than 80%, the virus is harvested to obtain porcine...

Embodiment 3

[0029] A culture method for improving porcine epidemic diarrhea virus virulence, comprising the following steps:

[0030] (1) The Vero cells were passaged in bottles at a volume ratio of 1:4, and then inserted into 1600 mL of DMEM medium containing 12vt% newborn bovine serum and a pH value of 7.4, and cultured at 37.5°C and 11r / h for 50h. A dense monolayer was obtained;

[0031] (2) pour out the used DMEM medium, then inoculate the porcine epidemic diarrhea virus of 3vt% in the dense monolayer of step (1) gained, cultivate 70min under the condition of 37.5 ℃ of temperature, 11r / h rotating speed, obtain mixture ;

[0032] (3) Add 1600mL of DMEM medium containing 3vt% newborn bovine serum, 12μg / mL insulin and 9g / L trehalose, and a pH value of 7.4 to the mixture obtained in step (2), at a temperature of 37.5°C and a rotational speed of 11r / h Under cultivation, when the lesion rate is greater than 80%, the virus is harvested to obtain porcine epidemic diarrhea virus.

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Abstract

The invention discloses a cultivation method for increasing the virulence of porcine epidemic diarrhea virus, which comprises the following steps: dividing Vero cells into bottles for passage, then inserting the cell growth solution containing neonatal bovine serum, and obtaining a dense monolayer after cultivation; Inoculate porcine epidemic diarrhea virus in a dense monolayer, and cultivate the mixture; add maintenance solution containing newborn bovine serum, insulin and trehalose to the mixture, rotate the culture, harvest the virus when the lesion rate is greater than 80%, and obtain porcine epidemic diarrhea Virus. The invention can significantly increase the virus content, avoid virus inactivation, and effectively solve the problem of low virus price.

Description

technical field [0001] The invention relates to the technical field of porcine virus cultivation, in particular to a cultivation method for increasing the virulence of porcine epidemic diarrhea virus. Background technique [0002] Porcine Epidemic Diarrhea Virus (PEDV) belongs to class Ⅰ coronavirus, which causes porcine epidemic diarrhea disease. Piglets infected with this virus have a high mortality rate. The morphological characteristics of PEDV in intestinal epithelial cells are the same as those of other coronaviruses, and the virus is assembled by budding through the endoplasmic membrane. However, the virus particles detected in fecal samples are pleomorphic and tend to be spherical, with a diameter of about 95-190nm (including fibrils). Most virions have an electron-opaque central region with swollen fibrils 18-23 nm long, arranged radially outward from the nucleocapsid. When producing the virus liquid antigen of porcine epidemic diarrhea virus (PEDV), although it c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12N5/071C12R1/93
CPCC12N5/0686C12N7/00C12N2500/34C12N2501/33C12N2770/20051
Inventor 肖昌建张传明邝声耀阴文奇周远成王子健
Owner SICHUAN HUASHEN ANIMAL BIOLOGICAL PRODS
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